This invention relates to the field of atherosclerosis. In particular, this invention pertains to the identification of a class of peptides that are orally administrable and that ameliorate one or more symptoms of atherosclerosis.
Cardiovascular disease is a leading cause of morbidity and mortality, particularly in the United States and in Western European countries. Several causative factors are implicated in the development of cardiovascular disease including hereditary predisposition to the disease, gender, lifestyle factors such as smoking and diet, age, hypertension, and hyperlipidemia, including hypercholesterolemia. Several of these factors, particularly hyperlipidemia and hypercholesteremia (high blood cholesterol concentrations) provide a significant risk factor associated with atherosclerosis.
Cholesterol is present in the blood as free and esterified cholesterol within lipoprotein particles, commonly known as chylomicrons, very low density lipoproteins (VLDLs), low density lipoproteins (LDLs), and high density lipoproteins (HDLs). Concentration of total cholesterol in the blood is influenced by (1) absorption of cholesterol from the digestive tract, (2) synthesis of cholesterol from dietary constituents such as carbohydrates, proteins, fats and ethanol, and (3) removal of cholesterol from blood by tissues, especially the liver, and subsequent conversion of the cholesterol to bile acids, steroid hormones, and biliary cholesterol.
Maintenance of blood cholesterol concentrations is influenced by both genetic and environmental factors. Genetic factors include concentration of rate-limiting enzymes in cholesterol biosynthesis, concentration of receptors for low density lipoproteins in the liver, concentration of rate-limiting enzymes for conversion of cholesterols bile acids, rates of synthesis and secretion of lipoproteins and gender of person. Environmental factors influencing the hemostasis of blood cholesterol concentration in humans include dietary composition, incidence of smoking, physical activity, and use of a variety of pharmaceutical agents. Dietary variables include amount and type of fat (saturated and polyunsaturated fatty acids), amount of cholesterol, amount and type of fiber, and perhaps amounts of vitamins such as vitamin C and D and minerals such as calcium.
Epidemiological studies show an inverse correlation of high density lipoprotein (HDL) and apolipoprotein (apo) A-I levels with the occurrence of atherosclerotic events (Wilson et al. (1988) Arteriosclerosis 8: 737-741). Injection of HDL into rabbits fed an atherogenic diet has been shown to inhibit atherosclerotic lesion formation (Badimon et al. (1990) J. Clin. Invest. 85: 1234-1241).
Human apo A-I has been a subject of intense study because of its anti-atherogenic properties. Exchangeable apolipoproteins, including apo A-I, possess lipid-associating domains (Brouillette and Anantharamaiah (1995) Biochim. Biophys. Acta 1256:103-129; Segrest et al. (1974) FEBS Lett. 38: :247-253). Apo A-I has been postulated to possess eight tandem repeating 22 mer sequences, most of which have the potential to form class A amphipathic helical structures (Segrest et al. (1974) FEBS Lett. 38: 247-253). Characteristics of the class A amphipathic helix include the presence of positively charged residues at the polar-nonpolar interface and negatively charged residues at the center of the polar face (Segrest et al. (1974) FEBS Lett. 38: 247-253; Segrest et al. (1990) Proteins: Structure, Function, and Genetics 8: 103-117). Apo A-I has been shown to strongly associate with phospholipids to form complexes and to promote cholesterol efflux from cholesterol-enriched cells. The delivery and maintenance of serum levels of apo A-I to effectively mitigate one or more symptoms of atherosclerosis has heretofore proven elusive.
This invention provides novel peptides administration of which mitigate one or more symptoms of atherosclerosis. In particular, it was a discovery of this invention that peptides comprising a class A amphipathic helix when formulated with xe2x80x9cDxe2x80x9d amino acid residue(s) and/or having protected amino and carboxyl termini can be orally administered to an organism, are readily taken up and delivered to the serum, and are effective to mitigate one or more symptoms of atherosclerosis.
Thus, in one embodiment, this invention provides a peptide that ameliorates a symptom of atherosclerosis, where the peptide ranges in length from about 10 to about 30 amino acids, comprises at least one class A amphipathic helix, comprises at least one xe2x80x9cDxe2x80x9d amino acid residue, protects a phospholipid against oxidation by an oxidizing agent, and is not the D-18A peptide (e.g. D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F (SEQ ID NO: 1) having all D form amino acid residues). In particularly preferred embodiments, the peptide further comprises a protecting group coupled to the amino and/or carboxyl terminus. Preferred protecting groups include, but are not limited to acetyl, amide, and 3 to 20 carbon alkyl groups, Fmoc, Tboc, 9-fluoreneacetyl group, 1-fluorenecarboxylic group, 9-fluorenecarboxylic group, 9-fluorenone-1-carboxylic group, benzyloxycarbonyl, Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl (Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr), Mesitylene-2-sulphonyl (Mts), 4,4-dimethoxybenzhydryl (Mbh), Tosyl (Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc), 4-methylbenzyl (MeBzl), 4-methoxybenzyl (MeOBzl), Benzyloxy (BzlO), Benzyl (Bzl), Benzoyl, (Bz), 3-nitro-2-pyridinesulphenyl (Npys), 1-(4,4-dimethyl-2,6-diaxocyclohexylidene)ethyl (Dde), 2,6-dichlorobenzyl (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-Cl-Z), 2-bromobenzyloxycarbonyl (2-Br-Z), Benzyloxymethyl (Bom), t-butoxycarbonyl (Boc), cyclohexyloxy (cHxO), t-butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl (tBu), Acetyl (Ac), and Trifluoroacetyl (TFA). In certain particularly preferred embodiments the peptide further comprises a first protecting group coupled to the amino terminus and a second protecting group coupled to the carboxyl terminus. Particularly preferred peptides comprise greater than about 50% amino acid sequence identity with human or mouse apo A-I1 or with the polypeptide encoded by the. exon encoding a class A amphipathic helix of human or mouse apo A-I1. In certain preferred embodiments, at least 50%, more preferably at least 75%, and most preferably at least 90% and even 100% of the enantiomeric amino acids are xe2x80x9cDxe2x80x9d amino acids. The peptide may be combined with a pharmacologically acceptable excipient (e.g. an excipient suitable for oral administration to a mammal).
In certain particularly preferred embodiments, the peptide comprises one or more of the following amino acid sequences: D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F (SEQ ID NO: 2), D-W-F-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F (SEQ-ID-NO:3), D-W-L-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F (SEQ-ID-NO:4), D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F(SEQ-ID-NO:5), D-W-L-K-A-F-Y-D-K-F-F-E-K-F-K-E-F-F (SEQ-ID-NO:6), D-W-F-K-A-F-Y-D-K-F-F-E-K-F-K-E-F-F (SEQ-ID-NO:7), D-W-L-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F (SEQ-ID-NO:8), D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-F-F (SEQ-ID-NO:9), D-W-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-A-F (SEQ-ID-NO:10), D-W-L-K-A-F-Y-D-K-V-F-E-K-L-K-E-F-F (SEQ-ID-NO:11), D-W-L-K-A-F-Y-D-K-V-A-E-K-F-K-E-F-F (SEQ-ID-NO:12), D-W-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-F-F (SEQ-ID-NO:13), E-W-L-K-L-F-Y-E-K-V-L-E-K-F-K-E-A-F (SEQ-ID-NO:14), E-W-L-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F (SEQ-ID-NO:15), E-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-F-F (SEQ-ID-NO:16), E-W-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-A-F (SEQ-ID-NO:17), E-W-L-K-A-F-Y-D-K-V-F-E-K-L-K-E-F-F (SEQ-ID-NO:18), E-W-L-K-A-F-Y-D-K-V-A-E-K-F-K-E-F-F (SEQ-ID-NO:19), E-W-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-F-F (SEQ ID NO:20), A-F-Y-D-K-V-A-E-K-L-K-E-A-F (SEQ ID NO:21), A-F-Y-D-K-V-A-E-K-F-K-E-A-F (SEQ ID NO:22), A-F-Y-D-K-V-A-E-K-F-K-E-A-F (SEQ ID NO:23), A-F-Y-D-K-F-F-E-K-F-K-E-F-F (SEQ ID NO:24), A-F-Y-D-K-F-F-E-K-F-K-E-F-F (SEQ ID NO:25), A-F-Y-D-K-V-A-E-K-F-K-E-A-F (SEQ ID NO:26), A-F-Y-D-K-V-A-E-K-L-K-E-F-F (SEQ ID NO:27), A-F-Y-D-K-V-F-E-K-F-K-E-A-F (SEQ ID NO:28), A-F-Y-D -K-V-F-E-K-L-K-E-F-F (SEQ ID NO:29), A-F-Y-D-K-V-A-E-K-F-K-E-F-F (SEQ ID NO:30), K-A-F-Y-D-K-V-F-E-K-F-K-E-F (SEQ ID NO:31), L-F-Y-E-K-V-L-E-K-F-K -E-A-F (SEQ ID NO:32), A-F-Y-D-K-V-A-E-K-F-K-E-A-F (SEQ ID NO:33), A-F-Y-D-K-V-A-E-K-L-K-E-F-F (SEQ ID NO:34), A-F-Y-D-K-V-F-E-K-F-K-E-A-F (SEQ ID NO:35), A-F-Y-D-K-V-F-E-K-L-K-E-F-F (SEQ ID NO:36), A-F-Y-D-K-V-A-E-K-F-K-E-F-F (SEQ ID NO:37), A-F-Y-D-K-V-F-E-K-F-K-E-F-F (SEQ ID NO:38), and/or the above sequences comprising amino acid analogs. The enantiomeric amino acids of such sequences preferably comprise at least one xe2x80x9cDxe2x80x9d amino acid. In certain preferred embodiments, at least 50%, more preferably at least 75%, and most preferably at least 90% and even 100% of the enantiomeric amino acids are xe2x80x9cDxe2x80x9d amino acids as described herein.
Such peptides can also include a protecting group (e.g., amide, acetyl, propionyl, and a 3 to 20 carbon alkyl, etc.) coupled to the amino or carboxyl terminus. In certain embodiments, the protecting group coupled to the carboxyl terminus is an amide. In certain embodiments, the protecting group coupled to the amino terminus is an acetyl, a propeonyl, or a 3 to 20 carbon alkyl. Certain peptides comprise both a carboxyl- and an amino-terminus protecting group. In one such embodiment, the amino terminus protecting group is a protecting group selected from the group consisting of acetyl, propionyl, and a 3 to 20 carbon alkyl; and the carboxyl terminal protecting group is an amide.
In certain embodiments, the peptide is one that protects a phospholipid against oxidation by an oxidizing agent selected from the group consisting of hydrogen peroxide, 13(S)-HPODE, 15(S)-HPETE, HPODE, HPETE, HODE, and HETE. The phospholipid can be a phospholipid selected from the group consisting of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (Ox-PAPC), 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (POVPC), 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (PGPC), 1-palmitoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine (PEIPC), 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (SAPC), 1-stearoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (SOVPC), 1-stearoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (SGPC), 1-stearoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine (SEIPC), 1-stearoyl-2-arachidonyl-sn-glycero-3-phosphorylethanolamine (Ox-SAPE), 1-stearoyl-2-oxovaleroyl-sn-glycero-3-phosphorylethanolamine (SOVPE), 1-stearoyl-2-glutaroyl-sn-glycero-3-phosphorylethanolamine (SGPE), and 1-stearoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylethanolamine(SEI PE).
In another embodiment, this invention provides a composition, suitable for oral administration, that ameliorates a symptom of atherosclerosis. The composition comprises a peptide that is a human apo A-I peptide or fragment thereof comprising a class A amphipathic helix, or an analogue of a human apo A-I peptide wherein said peptide has a first protecting group attached to an amino terminal and a second protecting group attached to a carboxyl terminal and further wherein said peptide comprises a plurality of D amino acid residues. The protecting groups include, but are not limited to the protecting groups described herein. In certain embodiments, more than half, more preferably more than 80%, and most preferably more than 90% or even all of the enantiomeric amino acids comprising the peptide are D amino acids. The composition can further comprise a pharmaceutically acceptable excipient (e.g., an excipient suitable for oral administration or an excipient suitable for injection). Preferred peptides are capable of protecting a phospholipid (e.g., 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (Ox-PAPC), 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (POVPC), 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (PGPC), 1-palmitoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine (PEIPC), 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (SAPC), 1-stearoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (SOVPC), 1-stearoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (SGPC), 1-stearoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine (SEIPC), 1-stearoyl-2-arachidonyl-sn-glycero-3-phosphorylethanolaamine (Ox-SAPE), 1-stearoyl-2-oxovaleroyl-sn-glycero-3-phosphorylethanolamine (SOVPE), 1-stearoyl-2-glutaroyl-sn-glycero-3-phosphorylethanolamine (SGPE), and 1-stearoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylethanolamine (SEI PE), etc.) from oxidization by an oxidizing agent (e.g. hydrogen peroxide, 13(S)-HPODE, 15(S)-HPETE, HPODE, HPETE, HODE, and HETE).
This invention also provides methods of ameliorating a symptom of atherosclerosis. The methods comprise administering to an organism (e.g. human or non-human mammal) one or more of the peptides described herein. In particularly preferred embodiments, such peptides comprise a plurality of xe2x80x9cDxe2x80x9d amino acids and/or are protected as described herein. The peptide is preferably orally administered to the organism and the organism is preferably an organism diagnosed as having or as at risk for one or more symptoms of atherosclerosis. In certain embodiments, the peptide can be provided as an isolated peptide or combined with a pharmacological excipient as described herein. The administration is preferably at a dosage sufficient to ameliorate one or more symptoms of atherosclerosis and/or to significantly reduce the likelihood of occurrence of one or more symptoms of atherosclerosis.
In still another embodiment, this invention provides a kit for ameliorating a symptom of atherosclerosis. Preferred kits include a container containing one or more of the peptides described herein. The peptides preferably comprise a plurality of xe2x80x9cDxe2x80x9d amino acids and/or are protected as described herein. In certain embodiments, the kit can optionally further include a pharmaceutically acceptable excipient and/or the peptide is provided combined with a with a pharmaceutically acceptable excipient (e.g. in a unit dosage formulation). Preferred kits provided the peptide(s) as a unit dosage formulation is for oral administration. The kits also, optionally, include instructional materials teaching the use of said peptide for ameliorating one or more symptoms of atherosclerosis and/or for reducing the likelihood of occurrence of one or more symptoms of atherosclerosis.
In certain embodiments, this invention excludes any one or more peptides disclosed in U.S. Pat. No. 3,767,040 and/or in Garber et al. (1992) Arteriosclerosis and Thrombosis, 12: 886-894. In preferred embodiments, this invention excludes peptides having the formula A1-B1-B2-C1-D-B3-B4-A2-C2-B5-B6-A3-C3-B7-C4-A4-B8-B9 where A1, A2, A3 and A4 are independently aspartic acid or glutamic acid, or homologues or analogues thereof; B1, B2, B3, B4, B5, B6, B7, B8 and B9 are independently tryptophan, phenylalanine, alanine, leucine, tyrosine, isoleucine, valine or xcex1-naphthylalanine, or homologues or analogues thereof; C1, C2, C3 and C4 are independently lysine or arginine, and D is serine, threonine, alanine, glycine, histidine, or homologues or analogues thereof; provided that, when A1 and A2 are aspartic acid, A3 and A4 are glutamic acid, B2 and B9 are leucine, B3 and B7 are phenylalanine, B4 is tyrosine, B5 is valine, B6, B8, and D are alanine, and C1, C2, C3 and C4 are lysine, B1 is not tryptophan.
Definitions
The terms xe2x80x9cpolypeptidexe2x80x9d, xe2x80x9cpeptidexe2x80x9d and xe2x80x9cproteinxe2x80x9d are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
The term xe2x80x9cclass A amphipathic helixxe2x80x9d refers to a protein structure that forms an xcex1-helix producing a segregation of a polar and nonpolar faces with the positively charged residues residing at the polar-nonpolar interface and the negatively charged residues residing at the center of the polar face (see, e.g., xe2x80x9cSegrest et al. (1990) Proteins: Structure, Function, and Genetics 8: 103-117).
The term xe2x80x9camelioratingxe2x80x9d when used with respect to xe2x80x9cameliorating one or more symptoms of atherosclerosisxe2x80x9d refers to a reduction, prevention, or elimination of one or more symptoms characteristic of atherosclerosis and/or associated pathologies. Such a reduction includes, but is not limited to a reduction or elimination of oxidized phospholipids, a reduction in atherosclerotic plaque formation and rupture, a reduction in clinical events such as heart attack, angina, or stroke, a decrease in hypertension, a decrease in inflammatory protein biosynthesis, reduction in plasma cholesterol, and the like.
The term xe2x80x9cenantiomeric amino acidsxe2x80x9d refers to amino acids that can exist in at least two forms that are nonsuperimposable mirror images of each other. Most amino acids (except glycine) are enantiomeric and exist in a so-called L-form (L amino acid) or D-form (D amino acid). Most naturally occurring amino acids are xe2x80x9cLxe2x80x9d amino acids. The terms xe2x80x9cD amino acidxe2x80x9d and xe2x80x9cL amino acidxe2x80x9d are used to refer to absolute configuration of the amino acid, rather than a particular direction of rotation of plane-polarized light. The usage herein is consistent with standard usage by those of skill in the art.
The term xe2x80x9cprotecting groupxe2x80x9d refers to a chemical group that, when attached to a functional group in an amino acid (e.g. a side chain, an alpha amino group, an alpha carboxyl group, etc.) blocks or masks the properties of that functional group. Amino protecting groups include, but are not limited to acetyl, or amino groups. Other amino protecting groups include, but are not limited to alkyl chains as in fatty acids, propionyl, formyl and others. Preferred carboxyl protecting groups include, but are not limited to groups that form amides or esters.
The phrase xe2x80x9cprotect a phospholipid from oxidation by an oxidizing agentxe2x80x9d refers to the ability of a compound to reduce the rate of oxidation of a phospholipid (or the amount of oxidized phospholipid produced) when that phospholipid is contacted with an oxidizing agent (e.g. hydrogen peroxide, 13(S)-HPODE, 15(S)-BPETE, HPODE, HPETE, HODE, HETE, etc.).
The terms xe2x80x9clow density lipoproteinxe2x80x9d or xe2x80x9cLDLxe2x80x9d is defined in accordance with common usage of those of skill in the art. Generally, LDL refers to the lipid-protein complex which when isolated by ultracentrifugation is found in the density range d=1.019 to d=1.063.
The terms xe2x80x9chigh density lipoproteinxe2x80x9d or xe2x80x9cHDLxe2x80x9d is defined in accordance with common usage of those of skill in the art. Generally xe2x80x9cHDLxe2x80x9d refers to a lipid-protein complex which when isolated by ultracentrifugation is found in the density range of d=1.063 to d=1.21.
The term xe2x80x9cGroup I HDLxe2x80x9d refers to a high density lipoprotein or components thereof (e.g. apo A-I, paraoxonase, platelet activating factor acetylhydrolase, etc.) that reduce oxidized lipids (e.g. in low density lipoproteins) or that protect oxidized lipids from oxidation by oxidizing agents.
The term xe2x80x9cGroup II HDLxe2x80x9d refers to an HDL that offers reduced activity or no activity in protecting lipids from oxidation or in repairing (e.g. reducing) oxidized lipids.
The term xe2x80x9cHDL componentxe2x80x9d refers to a component (e.g. molecules) that comprises a high density lipoprotein (HDL). Assays for HDL that protect lipids from oxidation or that repair (e.g. reduce oxidized lipids) also include assays for components of HDL (e.g. apo A-I, paraoxonase, platelet activating factor acetylhydrolase, etc.) that display such activity.
The term xe2x80x9chuman apo A-I peptidexe2x80x9d refers to a full-length human apo A-I peptide or to a fragment or domain thereof comprising a class A amphipathic helix.
A xe2x80x9cmonocytic reactionxe2x80x9d as used herein refers to monocyte activity characteristic of the xe2x80x9cinflammatory responsexe2x80x9d associated with atherosclerotic plaque formation. The monocytic reaction is characterized by monocyte adhesion to cells of the vascular wall (e.g. cells of the vascular endothelium), and/or chemotaxis into the subendothelial space, and/or differentiation of monocytes into macrophages.
The term xe2x80x9cabsence of changexe2x80x9d when referring to the amount of oxidized phospholipid refers to the lack of a detectable change, more preferably the lack of a statistically significant change (e.g. at least at the 85%, preferably at least at the 90%, more preferably at least at the 95%, and most preferably at least at the 98% or 99% confidence level). The absence of a detectable change can also refer to assays in which oxidized phospholipid level changes, but not as much as in the absence of the protein(s) described herein or with reference to other positive or negative controls.
The following abbreviations are used herein: PAPC: L-xcex1-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine; POVPC: 1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine; PGPC: 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine; PEIPC: 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phsophocholine; ChC18:2: cholesteryl linoleate; ChC18:2-OOH: cholesteryl linoleate hydroperoxide; DMPC: 1,2-ditetradecanoyl-rac-glycerol-3-phosphocholine; PON: paraoxonase; HPF: Standardized high power field; PAPC: L-xcex1-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine; POVPC: 1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine; PGPC: 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine; PEIPC: 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine; PON: paraoxonase; HPF: Standardized high power field; BL/6: C57BL/6J; C3H:C3H/HeJ.
The term xe2x80x9cconservative substitutionxe2x80x9d is used in reference to proteins or peptides to reflect amino acid substitutions that do not substantially alter the activity (specificity or binding affinity) of the molecule. Typically conservative amino acid substitutions involve substitution one amino acid for another amino acid with similar chemical properties (e.g. charge or hydrophobicity). The following six groups each contain amino acids that are typical conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
The terms xe2x80x9cidenticalxe2x80x9d or percent xe2x80x9cidentity,xe2x80x9d in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. With respect to the peptides of this invention sequence identity is determined over the full length of the peptide.
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., supra).
One example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle (1987) J. Mol. Evol. 35:351-360. The method used is similar to the method described by Higgins and Sharp (1989) CABIOS 5: 151-153. The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments. The program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters. For example, a reference sequence can be compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.
Another example of algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always  greater than 0) and N (penalty score for mismatching residues; always  less than 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=xe2x88x924, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).
In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA, 90: 5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
The term xe2x80x9cD-18A peptidexe2x80x9d refers to a peptide having the sequence: D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F (SEQ ID NO: 1) where all of the enantiomeric amino acids are D form amino acids.